Non-specific uptake of 18F-FAPI-04 in the pancreas and its related factors: a post-hoc analysis of an ongoing prospective clinical trial

This study aimed to analyze the characteristics of the non-specific uptake (NSU) of 18F-labeled fibroblast activation protein inhibitor (18F-FAPI) of the pancreas and investigate the related factors. Totally, 78 patients who underwent both 18F-fluorodeoxyglucose (FDG) and 18F-FAPI PET/CT examinations were divided into normal (n = 53) and NSU (n = 25) groups. The differences in general information, medical history, laboratory indexes and uptake were compared. Receiver operating characteristic (ROC) curves were used to analyze the optimal cut-off values. The correlations between 18F-FAPI-SUVmax and blood cell analysis, liver function indexes, tumor markers, and inflammatory indices were analyzed. The logistic regression model was used to estimate the independent factors. Both 18F-FAPI (4.48 ± 0.98 vs. 2.01 ± 0.53, t = 11.718, P < 0.05) and 18F-FDG (2.23 ± 0.42 vs. 2.02 ± 0.44, t = 2.036, P = 0.045) showed significantly higher in NSU group. Patients in the NSU group tended to be complicated with a history of drinking (P = 0.034), chronic liver diseases (P = 0.006), and surgery of gastrectomy (P = 0.004). ROC analysis showed cutoff values of 3.25 and 2.05 for 18F-FAPI and 18F-FDG in identifying the NSU. Patients in the NSU group showed less platelet count, higher platelet volume, higher total bilirubin, direct or indirect bilirubin (P < 0.05). Platelet count, platelet crit, large platelet ratio, aspartate aminotransferase (AST), α-l-fucosidase, and total, direct or indirect bilirubin were correlated with 18F-FAPI-SUVmax (P < 0.05). AST [1.099 (1.014, 1.192), P = 0.021] and total bilirubin [1.137 (1.035, 1.249), P = 0.007] were two independent factors in the step forward logistic regression, and platelet/% [1.079 (1.004, 1.160), P = 0.039] and total bilirubin [1.459 (1.016, 2.095), P = 0.041] were two independent factors in the step backward logistic regression for the prediction of pancreatic uptake of 18F-FAPI. 18F-FAPI-PET/CT was better than 18F-FDG in predicting the pancreatic NSU, and NSU is related to a history of drinking, chronic liver diseases, gastrectomy, heteromorphic platelet, and impaired liver function.

Fibroblast activation protein (FAP), a type II transmembrane glycoprotein, is an antigenic molecule on the surface of cancer-associated fibroblasts (CAFs) 1,2 .FAP is often highly expressed in tumors of epithelial origin 3 , and FAP inhibitors (FAPIs) are small molecule compounds synthesized based on the structure of quinoline, which can bind specifically to FAP on the surface of CAFs.Thus, FAPI positron emission tomography/computed tomography (PET/CT) is commonly used in the clinical imaging of malignancies.Besides, FAP can also be found during the remodeling of the extracellular matrix, such as chronic inflammation, arthritis, fibrosis, and ischemic heart tissue after a myocardial infarction 4 .Thus, non-oncological FAPI uptake can be seen in liver fibrosis, cirrhosis, arthritis, cardiovascular disease, IgG4-related disease, and benign tumors 5 .The pancreas, a vital digestive organ, is surrounded by a fibrous capsule from which connective tissue septa extend into the gland.FAPI is highly expressed in pancreatic cancers due to the abundant extracellular matrix and stromal cells, including CAFs.Although mesenchyme accounts for approximately 15-25% of total pancreas volume 6 , FAPI uptake was low in the normal tissues of the pancreas due to the inactivated mesenchyme fibroblast, with a maximum standardized uptake value (SUVmax) of 1.04-3.0,even lower than the FDG uptake [7][8][9] .
Except for the oncological uptake, FAPI accumulation has also been confirmed in acute pancreatitis (average SUVmax: 7.5 ± 3.5, n = 8) 10 and IgG4-related pancreatitis (average SUVmax: 15.2 ± 9.0, n = 19) 11 .Zhang 12 has reported 7 cases of focally elevated uptake of 68 Ga-FAPI-04 in the pancreas, and lesions were identified, including pancreatic pseudocysts, sites of prior pancreatitis, and IgG 4-related disease.However, in our clinical observation, we noticed that some cases without pancreatic cancer and acute or IgG4-related pancreatitis also showed an accumulation of 18 F-FAPI in the pancreas.Rare studies have mentioned this non-specific uptake (NSU) of FAPI in the pancreas.Thus, this study aimed to analyze the characteristics of the NSU of 18 F-FAPI of the pancreas and investigate the potential independent factors.

Materials
This was a post-hoc analysis of a single-center, ongoing prospective clinical trial conducted at the First Affiliated Hospital of Xi'an Jiaotong University (approval number: XJTU1AF-CRF-2022-021).The study protocol was approved by the Ethics Committee of Xi'an Jiaotong University (XJTU1AF2022LSL-021).This study was registered at ClinicalTrials Gov (number NCT05788874).All participants signed an informed consent form following the national regulations of the Declaration of Helsinki and Good Clinical Practice.
This retrospective study included 86 patients who underwent both 18 F-FDG and 18 F-FAPI PET/CT examinations at an interval of 1-43 days.One patient with a history of pancreatic resection was excluded, and all patients were followed up for the final diagnosis.4 cases of autoimmune pancreatitis (AIP) and 3 cases of pancreatic cancer (PC) confirmed by imaging manifestations, or histopathological evidence, were excluded.None of the women included in this study were pregnant.The study flow is displayed in Fig. 1.

PET/CT image acquisition
Preparation of 18 F-FDG and 18 F-FAPI-04. 18F-FDG and 18 F-FAPI-04 were synthesized using a GE MINItrace cyclotron and Tracerlab FX-FN multifunctional synthesizer.The synthetic precursor kits, FDG and FAPI, were purchased from ABX (Germany) and Jiangyuan (China).The radio-chemical purity of the synthesized drugs exceeded 95%.Further physical and biological quality controls were performed in compliance with current pharmacopeias.
PET/CT imaging.All patients underwent PET/CT on the same scanner (Gemini TF PET/CT, Philips, Netherlands).For 18 F-FDG PET/CT scans, the patients fasted for ≥ 6 h, and the blood glucose levels were ≤ 12 mmol/L before the injection (370 MBq/kg).There was no special preparation for patients on the day of 18 F-FAPI PET/CT scanning, and 18 F-FAPI PET/CT scan was initiated 60 min after injection (370 MBq/kg).
Images encompassing the head to the mid-thigh were obtained for both scans.PET scans (1.5 min/bed position, 6-10 bed/per person) were acquired with an interval of 68.5 ± 12.1 min (range, 47 ~ 90 min) in threedimensional mode after the injection.CT scans were performed (tube voltage 120 kV, tube current 50-100 mAs, layer thickness, 5 mm; 512 × 512 matrix) for attenuation correction and anatomical reference.The images were then reconstructed using an iterative method and time of flight, and the image data were transferred to an Extended Brilliance Workstation for image interpretation.

PET/CT image evaluation
The 18 F-FDG and 18 F-FAPI PET/CT images were interpreted by two > 10 years of experienced PET/CT physicians (Y.L.) and (C.S.), and a consensus was performed.Any disagreements were resolved by a more experienced PET/CT physician.The NSU group was defined as the increased radioactivity uptake of FAPI in the pancreas compared to the background, without confirming pancreatic cancer, acute or IgG4-related pancreatitis by other imaging methods, serology, or pathological results (if available).
For the normal group, the criteria were: (1) without 18 F-FAPI uptake compared with the background; (2) without elevated serum tumor biomarkers or pancreatitis biomarkers; (3) without a history of pancreatic disease; or (4) without pancreatic-related symptoms or body signs.
The same nuclear medicine physician (C.S.) measured the pancreas' SUVmax values using circular regions of interest (ROI) placed on axial slices.The CT image features of NSU patients, including pancreatic swelling, calcification, and irregular dilatation of the pancreatic ducts, were also evaluated.

Statistical analysis IBM SPSS Statistics for
Windows, version 25.0 (IBM Corp., Armonk, NY, USA) was used for the statistical analyses.Continuous variables with normal distribution and variance uniformity were represented as means ± standard deviation and analyzed by independent-sample t-tests, and continuous variables without normal distribution were expressed as median [25%, 75%] and were tested using the Mann-Whitney U test.Categorical variables were defined as several cases and analyzed by χ 2 or Fisher exact tests.Kappa test was used for consistency evaluation between the two readers.
Receiver operating characteristic (ROC) curves were used to analyze the optimal cut-off value of SUVmax for predicting NSU of the pancreas [determined by Youden index = sensitivity + specificity − 1].The DeLong test was used to compare the performance between the 18 F-FAPI and 18 F-FDG using Medcalc (v22.009,MedCalc Software Ltd, Belgium).
The correlations between FAPI-SUVmax and blood cell analyzed indicators, liver function indicators, tumor markers, and inflammatory indices were analyzed by Pearson correlation (only continuous variables with normal distribution and variance uniformity) or Spearman correlation.The logistic regression model was used to estimate the factors related to pancreatic NSU.Statistical significance was set at P < 0.05.

Clinical characteristics
Seventy-eight patients (45 males, 33 females) were analyzed with a median age of 60 years old (25% ~ 75%, 52 ~ 68 years old).The primary malignancy or the primary diagnosis was shown in Supplementary Table 1.
Among the 78 patients included, 25 (32.05%)showed NSU in the pancreas with a pattern of diffused uptake.For the 25 cases in the NSU group, the agreement rate of the two readers was 84.00% (21/25), and 4 cases (with the SUVmax of 2.57, 3.33, 3.41 and 3.41, respectively) were judged by the 3rd reader.For the 53 cases in the normal group, the agreement rate of the two readers was 98.11% (52/53), and 1 cases (with the SUVmax of 3.17) were judged by the 3rd reader.The Kappa of the two readers was 0.848 (P < 0.001).None of the patients in the NSU patients showed pancreatic swelling and calcification, and only one patient (4%) showed irregular dilatation of the pancreatic ducts.The tracer uptake of 18 F-FAPI showed significantly higher in the NSU group than in the normal group (4.48 ± 0.98 vs. 2.01 ± 0.53, t = 11.718,P < 0.001).Meantime, although with the slight difference of SUVmax, the tracer uptake of FDG also showed significantly higher in the NSU group than that of in the normal group (2.23 ± 0.42 vs. 2.02 ± 0.44, t = 2.036, P = 0.045), see as Table 1.www.nature.com/scientificreports/ The tracer uptake in the normal group between the 18 F-FAPI and 18 F-FDG showed no difference (P = 0.890), while the tracer uptake in the NSU group between 18 F-FAPI and 18 F-FDG showed a significant difference (P < 0.001), see as Fig. 2.
In the Chi-Square test, patients in the NSU group weretended to complicate with a history of drinking (P = 0.034), chronic liver diseases (P = 0.006), and surgery of gastrectomy (P = 0.004), see Table 1, Figs. 3 and 4.

ROC analysis of SUVmax value of two tracers
ROC analysis showed that the cutoff value for FAPI-SUVmax to identify the NSU from normal was 3.25, with an area under the curve (AUC), sensitivity, and specificity of 0.992, 0.960, and 1.000, respectively, see Fig. 5.The cutoff value for FDG-SUVmax to identify the NSU from normal was 2.05, with an AUC an area under the curve (AUC), sensitivity, and specificity of 0.630, 0.720, and 0.509, respectively, see Table 2.The Delong test showed that the two ROC curves differed significantly (Z = 5.348, P < 0.001).

Comparisons of blood cell analysis, liver function indicators, tumor biomarkers, and inflammatory indices between two groups
Patients in the NSU group showed a lower level of platelet count (NSU vs. Normal, 182.26 ± 76.70 vs. 229.30± 74.17, t = 2.490, P = 0.015), higher level of mean platelet volume (NSU vs. Normal, 10.93 ± 1.34 vs.     3.The tumor biomarkers and the inflammatory indices didn't statistically differ in the two groups (P > 0.05), see Table 4.

Discussion
This study tried to analyze the characteristics of the NSU uptake of 18 F-FAPI in the pancreas by comparing it with the 18 F-FDG head-to head.In the previous studies, non-oncological FAPI uptake was mainly explained as the pancreatic pseudocysts, sites of prior pancreatitis, and IgG 4-related disease 12 .However, in our clinical observations, approximately one-third of patients (25/78) without evidence of AIP and PC showed a kind of diffused NSU FAPI uptake in the pancreas, which means the fibrotic or chronic inflammatory changes of the pancreatic tissue.The difference in SUVmax in the two groups (NSU vs. normal, 4.48 ± 0.98 vs. 2.01 ± 0.53, P < 0.05) was notable and can be identified by human eyes.Meantime, the difference of SUVmax for 18 F-FDG PET/CT scan in two groups (NSU vs. normal, 2.23 ± 0.42 vs. 2.02 ± 0.44, P = 0.045), although statistically significant, was slight and was challenging to identify by human eyes.In the ROC analysis, SUVmax derived from 18 F-FAPI PET/ CT scan showed higher AUC, sensitivity, specificity, and accuracy in differentiating the pancreatic NSU than SUVmax derived from 18 F-FDG (P < 0.05), and the Delong test of two ROC curves showed statistically different (Z = 5.348, P < 0.001).These results indicated that 18 F-FAPI can detect chronic or fibrotic pancreas lesions that were negative in 18 F-FDG.
In our cohort, the cutoff value of the normal pancreas was 3.25 for 18 F-FAPI and 2.05 for 18 F-FDG.Previous studies reported 18 F-FAPI-SUVmax for the normal pancreatic tissues varied from 1.04 to 3.0 7,9 .Frederik L et al. et al. 7 conducted a head-to-head intra-individual comparison of biodistribution of 68 Ga-FAPI and 18 F-FDG PET/ CT in cancer patients, and their results showed the pancreas uptake showed significantly lower for 68 Ga-FAPI compared with 18 F-FDG (1.82 vs. 1.99;P = 0.027).However, in our cohort, pancreas uptake showed no difference for 18 F-FAPI compared with 18 F-FDG (2.01 ± 0.53 vs 2.02 ± 0.44, P = 0.900).Cihan Gündoğan analyzed the 68 Ga-FAPI-04 uptake in healthy tissues, and the pancreas showed a median SUVmax of 2.61 (minimal-maximal, 1.04 ~ 2.58) 13 , which is very close to our results.
In our cohort, heavy drinking, chronic liver diseases, and surgery of gastrectomy showed significant correlations with higher 18 F-FAPI uptake.Previous study in a large-scale population shows that smoking, obesity, long-term drinking, diabetes, liver diseases, and long-term malnutrition are significantly associated with an increased risk of pancreatic disease 14 .Alcohol continues to be the an critical risk factor for chronic pancreatitis 15 .Therefore, alcoholism may have caused chronic pancreatitis.The surgery of gastrectomy, but not the intestine enterectomy, showed a correlation with pancreatic NSU, possibly due to the pancreatic fistula or biochemical leakage after gastrectomy 16 , which can lead to acute or chronic pancreatitis.
In our results, chronic liver disease, such as hepatitis or cirrhosis, but not liver metastasis, showed a correlation with pancreatic NSU.This can be explained.Hepatotropic viruses, such as hepatitis A, B, and E, were reported to affect of various extrahepatic tissues, such as the kidney, thyroid, pancreas, and bone marrow 17,18 .The presence of these viruses in pancreatic tissue induces fibrotic or chronic inflammatory changes resulting from the excessive deposition of the extracellular matrix, with the possibility of progression to metaplasia and subsequently, www.nature.com/scientificreports/ a done deal, 18 F-FAPI may be a new way to visible the potential fibrous changes in the pancreas, which is not shown by the traditional imaging modalities.
In the laboratory test, patients in the NSU group showed a lower platelet count, higher mean platelet volume, and large platelet ratio for the complete blood cells analysis (P < 0.05), which means worse platelet morphology.Previous studies showed that the increase of atypical platelets may predict worse clinical outcomes 21,22 and poor prognosis in patients with pancreatic cancer 23 .Also, patients in the NSU group showed higher levels of AST, total bilirubin, direct bilirubin, indirect bilirubin, and α-K-fucosidase in the serum (P < 0.05), which means impaired liver function or liver fibrosis in the patients with NSU of pancreas.In the logistic regression model, large platelet ratio, AST, and TBIL may be the independent factors for pancreatic 18 F-FAPI uptake.This can be explained, as AST and the platelet are regarded as the important biomarkers in the evaluation of the liver fibrosis 24,25 .
To our knowledge, this is the first reported set of pancreatic NSUs for the 18 F-FAPI.Whether there were NSU or not, the physician should carefully evaluate pancreatic uptake of 18 F-FAPI, integrate the medical history and laboratory tests, and follow up for the final diagnosis in clinical practice.
Our study has certain limitations.The first limitation is the limited sample size.Second, there was no histopathological confirmation due to no clear or clinical indication of puncture for the NSU FAPI uptake.Thus, the follow-up is necessary, including the abdominal symptoms, laboratory test, or histo-pathological evidence if available.
In conclusion, we reported about 1/3 pancreatic NSU of 18 F-FAPI, besides the AIP-or PC-related uptake.The NSU may be a result of a history of heavy drinking, chronic liver diseases, and surgery for gastrectomy.Patients with NSU showed lower levels of platelet count, larger platelet volume, and higher levels of AST, total bilirubin, direct bilirubin, indirect bilirubin, and α-L-fucosidase in the serum.Large platelet ratio, AST, and TBIL may be the independent factors for pancreatic 18 F-FAPI uptake.

Figure 3 .Figure 4 .
Figure 3. NSU in the18 F-FAPI after the surgery of gastrectomy and negative in the18 F-FDG PET/CT.A male patient, 55 years old, after surgery of gastrectomy for gastric cancer.18F-FDG PET/CT (A) showed no FDG concentration in the pancreas, and18 F-FAPI PET/CT imaging (B) showed a high concentration of diffuse FAPI in the pancreas, with an SUVmax of 5.3.FAPI fibroblast activation protein, FDG fluorodeoxyglucose, PET/CT positron emission tomography/computed tomography.SUVmax maximum standardized uptake value.

Table 1 .
Clinical characteristics of the patients in two groups.NSU non-specific uptake, BMI body mass index, FAPI fibroblast activation protein, FDG fluoro-deoxyglucose.

Table 3 .
Comparisons blood cells and liver function indexes in two groups.4patients in normal group and 1 patient in NSU group were missed in the blood cell analysis; 4 patients in normal group and 2 patients in NSU group were missed in the liver function analysis; 34 patients in normal group and 10 patients in NSU group were included in the total bile acids comparison; 48 patients in normal group and 21 patients in NSU group were included in the blood glucose comparison; NSU non-specific uptake, FAPI fibroblast activation protein, FDG fluoro-deoxyglucose, CV coefficients of variation, SD standard deviation, AST aspartate aminotransferase, ALT alanine transaminase, TBIL total bilirubin, DBIL direct bilirubin, IDBIL indirect bilirubin.a Z test.Vol:.(1234567890)Scientific Reports | (2024) 14:11141 | https://doi.org/10.1038/s41598-024-62005-2